Many biological techniques, including gene expression analyses such as hybridization assays, reverse transcription polymerase chain reaction (RT-PCR), cloning, restriction analysis, and sequencing use high-purity, intact RNA.
Several methods for isolating RNA are known which fall into two general categories, namely liquid phase and solid phase purification. In liquid phase isolation, the RNA remains in the liquid phase while impurities are removed by processes such as precipitation and/or centrifugation. In solid phase isolation, the RNA is bound to a solid support while impurities such as DNA, proteins and phospholipids are selectively eluted or do not bind at all to said solid support.
A widespread method of RNA isolation involves the use of chaotropic agents and organic solvents in order to bind the RNA to a solid phase, such as silica. Hereby, several organic solvents have been identified as suitable for this purpose such as alcohols (e.g. methanol, ethanol or isopropanol; see EP1 502 951 A1), ketones (e.g. diethylketone, methylethylketone, methylpropylketone, isobutylmethylketone; see EP 1 524 317 A1) or ethers such as diethylene glycole diethylether (see EP 1 529 841 A1). Most of the compounds are critical with regard to inflammability or toxicity or at least exhibit a very unpleasant odor. Furthermore, the RNA yields and the RNA integrity are often suboptimal and the DNA contamination is always a critical issue.
Hence, there is still a need for an improved methods and reagents for isolation of RNA.